Investigating The Influence of Native Extracellular Matrix on Neuromuscular Organoids Derived from Induced Pluripotent Stem Cells

The use of human induced pluripotent stem cell (hiPSC) technology, coupled with diverse culture methods, has enabled the generation of in vitro tissue/organ models that are reflect the genetic specificity of an individual. Moreover, hiPSC-based model overcome to the specie specificity response limitation given by other animal models. Commonly hiPSC-based studies primarily focused on the differentiation of hiPSCs into a single specific cell type, such as neurons or cardiomyocytes, which are than usually cultured in two-dimensional (2D) standard conditions. Recent advancements in biomaterial science have facilitated the development of three- dimensional (3D) culture methods, which can combine multiple cell types derived from hiPSCs and more closely mimic in vivo organ organization. A step forward has been recently achieved with the development of organoids, self-assembled 3D aggregates that present both stem and differentiated cells typical of a defined organ. Organoids can be derived from hiPSCs, enabling the modeling of complex tissue structures in a patient-specific fashion. Despite this, organoid field is on its infancy and several limitations, as scarce cytoarchitecture and cell type reproducibility, and poor cell maturation, still needs to be solved. Human neuromuscular organoids (NMOs) derived from hiPSCs represent a valuable tool for in vitro studies of functional human skeletal muscle (SkM). The multicellular interactions between the neural and muscular systems, which are crucial for SkM homeostasis and function, can be modeled and studied through these 3D in vitro systems. However, since NMO grow as spheroids the functional activity of the neuromuscular system is not really reproducing the structural/functional correlation observed in vivo. In this thesis, our first objective was to optimize human NMO cultures. Specifically, we developed an expansion protocol for NMOs to maximize sample production. Additionally, we characterized the cell populations present within the NMOs following single-cell isolation and evaluated the behavior of the NMOs following mechanical digestion. Our findings provide valuable insights into the production and the optimization of NMO expansion from hiPSCs, which is an essential step for scaling up experimental studies. With the aim to overcome the uncontrollable folding of the NMOs into a spheroidal 3D organization, here we implemented tissue-derived biomaterials to control NMO morphogenesis during hiPSCs differentiation. To do so, and in order to maintain tissue complexity, scaffolds were generated through decellularization process of native tissue. Decellularized extracellular matrix (dECM) in conjunction with the supplementation of small molecules that promote hiPSCs differentiation was adopted here as strategy to control NMO 3D architecture. Since greater neuromuscular system functionality and cell maturation was revealed in NMO derived in presence of dECM (tNMO), the subsequent objective of this thesis was to assess the contribution of mouse diaphragm-derived dECM in the differentiation of hiPSCs and the establishment of a functional neuromuscular system. Our observations revealed that dECM played a fundamental role in promoting the differentiation of hiPSCs into bipotent neuromesodermal progenitors (NMPs), capable of developing into both neural and muscular lineages. Moreover, the constructs obtained following 30 days of differentiation in the presence of dECM facilitated the establishment of a functional neuromuscular system capable of contracting in response to neurotransmitter stimulation. Overall, the results obtained in this thesis strongly support the concept that NMO derived from hiPSCs are a valuable tool for mimicking the human neuromuscular system in vitro. Finally, here we showed that the skeletal muscle ECM can directly influence hiPSCs commitment, resulting in the engineering of tNMOs with high levels of functionality and maturation.